THROMBOSIS RESEARCH 71; 139-148,1993

0049-3848/93

$6.00 + .OO Printed in the USA.

Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

SUPPRESSIVE

EFFECT OF HUMAN BLOOD COAGULATION

FACTOR XIII

ON THE VASCULAR

PERMEABILITY

INDUCED BY ANTI-GUINEA

PIG

ENDOTHELIAL

CELL ANTISERUM

IN GUINEA PIGS

Keizo Hirahara, Kazuhiko Shinbo, Mikiko Takahashi

and Tetsuro Matsuishi

Pharma Research Laboratories, Hoechst Japan Limited, l-3-2,

Minamidai, Kawagoe-shi, Saitama, 350 Japan

(Received 12.11 .1992; accepted in revised form 20.4.1993 by Editor A. Takada)

Abstract

We investigated the effect of blood coagulation factor

XIII(FXII1) on enhanced permeability induced by anti-

endothelial cell antiserum, that was produced by the

immunization

of guinea pig endothelial

cells with

adjuvant into rabbits repeatedly.

We have found that

this

antiserum

reacts

to

human

and

guinea

pig

endothelial cells but not guinea pig fibroblast cells.

The permeability was enhanced by intradermal injection

of 400-fold dilution of this antiserum into dorsal skin

of guinea

pigs.

The mixture

of equal volume

of

antiserum

and FXIII was intradermally

injected into

dorsal skin of guinea pig after Evans blue injection,

and 15 minutes later the quantity of Evans blue at the

each injection site was determined.

We recognized the

suppressive effect of FXIII on the dye leakage. We also

studied the suppressive effect on swelling induced by

the antiserum.

After the subcutaneous injection of the

mixture of antiserum and FXIII into the back of guinea

pigs,

we measured

the thickness

of skins

at the

injection site after day 1, 2 and 3. As a result, FXIII

significantly suppressed the swelling.

We found that

FXIII suppresses the acute and subacute permeability

enhancement.

These results suggest that FXIII plays an

important role on an inflammatory site and that it may

exert as an anti-inflammatory protein.

Blood

coagulation

factor XIII (FXIII),

Key words: Factor XIII, anti-endothelial

permeability, anti-inflammatory protein,

139

the

last

enzyme in the

cell antiserum, vascular

Schonlein Henoch purpura

140

PERMEABILITY

INHIBITION BY FXIII

Vol. 71, No. 2

blood coagulation cascade, is a transamidase that catalyzes the

formation of y - gultamyl - E: - lysyl peptide crosslinks between

polypeptide chains in adjacent fibrin monomers and other plasma

proteins(l,2,3).

Crosslinks

of each fibrin molecule caused a

marked increase in the rigidity of the clot network(4).

On the

other hands, the crosslinks between fibrin and cellular matrix

protein such as fibronectin may exert to connect fibrin molecules

with the injury sites(5).

It is well known that clots play an

important role in the prevention of further tissue damage and in

subsequent wound healing(6).

Schijnlein Henoch purpura (SHP) is

characterized

by hemorrhagic

skin lesions, abdominal

symptoms

including

gastro-intestinal

bleeding,

renal involvement

with

proteinuria and hematuria and swelling of joints(7).

The symptoms

are ascribed

to generalized

inflammation

of arterioles

and

capillaries.

That is, the local changes of the coagulation and

fibrinolytic

system due to immunoreaction

were induced in the

affected vessels.

In 1977, Henriksson and colleagues described a

lowering

of FXIII

activity

during

the acute phase

of this

disease(8).

The mechanism of the decrease of FXIII activity in

the acute phase of SHP has not yet been clarified.

Destruction of

FXIII molecules by protease derived from leukocytes which migrated

into the inflammation

sites has been proposed(9).

In this

connection,

Kamitsuji

and Fukui

et al. reported

that

the

administration

of FXIII

concentrate

may contribute

to the

improvement

of

gastro-intestinal

complications

of

SHP

patients(l0).

Recently FXIII concentrate (Fibrogammin P) is used

for the treatment of SHP patientstll).

According to Matsuoka (121,

Bowie et a1.(13) and Ito et a1.(14), this vasculitis of SHP is

regarded

as the immunovascular

disease that antibody-antigen

complexes on the vascular capillary endothelial cells enhances the

vascular permeability. Consequently non-thrombocytopenic

purpura

caused by the injection of anti-endothelial

cell antiserum(l5).

In the present study, we investigated whether or-not human FXIII

suppresses the enhancement of permeability and swelling induced by

anti-endothelial cell antiserum in guinea pigs.

MATERIALS AND METHODS

Materials

Materials were purchased from the following suppliers: Dulbecco

phosphate buffer, Dulbecco MEM, FCS(Gibco, USA), ECGS(Calbiochem,

USA), ^{Freund's adjuvant(Difco, USA), FITC conjugated anti-rabbit}

IgG(Cappe1,

USA), Evans blue, ^potassium

hydroxide(KOH,

Kanto

Kagaku, Japan), phosphoric acid(Wako Pure Chemical, Japan), Guinea

pig complement(Kyokuto,

Japan), and Human FXIII(Fibrogammin

P,

Behringwerke, FRG).

Vol. 71, No. 2

PERMEABILITY INHIBITION BY FXIII

141

Preparation of anti-guinea pig endothelial cell antiserum

Guinea pig endothelial cells were isolated from the main artery

and vena cava(l6), then cells were inoculated into tissue culture

dishes and incubated for several days with Dulbecco MEM containing

15% FCS and 37.5 pg/ml ECGS till reaching confluency. Confluent

monolayer was harvested by a cell scraper.

The cells were rinsed

twice with Dulbecco phosphate buffered saline(pH 7.2). These cells

were used as an antigen for the production of anti-endothelial

cell antiserum.

The antiserum was obtained from rabbits immunized

with emulsion

of Freund's

complete

adjuvant

with guinea pig

endothelial

cells,

and boosted

with

emulsion

of Freund's

incomplete

adjuvant.

After

several times of boosting,

the

antibody titer was measured with guinea pig endothelial cells by

the methods

of cytolysis

and

indirect

immunofluorescence

microscopy using FITC conjugated anti-rabbit IgG(17).

Measurement of antibody titer of anti-endothelial cell antiserum

Confluent monolayer of guinea pig endothelial cells in a 96-well

plate was incubated with 50 ~1 of variously diluted antiserum in

Dulbecco MEM-15% FCS for 30 min.

The medium was then replaced to

50 11 of 5% guinea pig complement in Dulbecco MEM-15% FCS and the

cells were further incubated for 30 min. After addition of 10 ~1

of trypan blue solution, the cell layers were photographed

to

evaluate the extent of cell lysis.

Indirect immunofluorescence

microscopy was done as follows. The antiserum was serially diluted

two times.

The diluted antiserum was then incubated with the main

artery at room temperature for 1 hour and rinsed 3 times with

Dulbecco phosphate buffer.

After washing, lOOO-fold dilution of

FITC conjugated

anti-rabbit

IgG was added to the sections,

incubated for 30 minutes at room temperature, and washed 3 times

with Dulbecco phosphate buffer.

All sections were observed by a

Nikon microscope equipped with a mercury lamp.

The titer was

taken as a highest dilution which gave a fluorescent staining just

above the background staining of normal serum controls.

Duration of activity of permeability enhancement

Measurement of permeability was studied according to Yamamoto et

al. (18) .

A 100 ~1 portion

of 50-fold diluted antiserum

was

intradermally

injected into the back of a guinea pig before

intravenous

injection of 0.5 ml of 1 % Evans blue.

After 15

minutes of the Evans blue injection, the back skins were harvested

and the blue lesions were observed.

Suppressive effect of FXIII on the permeability enhancement

A 100 ~1 portion of either each diluted antiserum or the mixture

of equal

volume

of FXIII

and the

diluted

antiserum

was

142

PERMEABILITY INHIBITION BY FXIII

Vol. 71, No. 2

intradermally injected into the dorsal skin of guinea pigs after

intravenous injection of Evans blue. After 15 minutes, skins were

harvested and blue lesions in the skins were observed.

Extraction of Evans blue from guinea pig skins

Evans blue was extracted from skins, soaked with 1 ml of 1 M KOH

solution, and incubated at 37'C overnight.

After the incubation,

3 ml of 0.6 N phosphoric acid and 3 ml of FRIGEN(Behringwerke,

FRG), ^{a defatting agent,} was added to each tube and mixed for 30

sec. with a Vortex mixer.

Each tube was centrifuged at 3000 rpm

for 15 minutes, and the absorbance of the supernatant was measured

at 620 nm(19).

Suppressive effect of FXIII on the swelling

One milliliter of equal volume mixture of FXIII and the intact

antiserum was subcutaneously

injected into the dorsum of guinea

pigs.

After days 1, 2 and 3, the skins were harvested and the

thickness was measured with a slide caliper at injection sites as

a marker of swelling.

The swelling was shown by the difference of

the thicknesses between a injection and a non-injection site.

RESULTS

Characterization

of polyclonal anti-guinea pig endothelial cell

antiserum

The antibody titer was determined with guinea pig endothelial

cells by the methods of cytolysis and indirect immunofluoresence

microscopy using FITC conjugated anti-rabbit IgG.

As a result,

the 50% cytolysis

was observed

by the 60-fold

dilution

of

antiserum, and the fluorescence was observed by 400-fold dilution.

The antiserum exhibited the reactivity with not only guinea pig

but also human endothelial cells.

However it did not react with

guinea pig fibroblasts.

When the cryosection of the main artery

of a guinea pig was used for the indirect immunofluorescence test,

the fluorescence

was observed on the inner membrane which was

seemed to be endothelial

cell.

It was also found that the

antiserum reacted with the extracellular matrix proteins produced

by endothelial cells(data not shown).

Enhanced permeability

First, we studied whether this antiserum induced the permeability

in guinea pigs.

The variation of permeability after intradermal

injection

is shown in Fig. 1.

The permeability

reached the

maximum

within

5 minutes.

This activity

for enhancing

the

permeability

almost disappeared

within

30 minutes

after the

injection.

This permeability enhancing phenomenon was classified

as a short lasting reaction.

We next investigated

the dose

response of this antiserum.

As shown in Fig. 2, the activity of

Vol. 71, No. 2

PERMEABILITY

INHIBITION

BY FXIII

143

Time course of permeability enhancement induced by anti

-endothelial

cell antiserum.

Antiserum was injected

into a guinea pig at varying times before intravenous

dye injection.

Time 0 means an intradermal injection

immediately

after intravenous

dye injection.

(A) :

antiserum,

60

min,

(B) :

antiserum,

30

min,

(C) :

antiserum,

0 min,

(D) :

saline,

30

min,

(E) :

rabbit

serum, 60

min, (F): rabbit serum, 30

min, (G): rabbit

serum, 0 min, (H): saline, 0 min

FIG. 1.

enhancing the permeability is recognized by 400-fold dilution of

antiserum.

The effect of FXIII was examined on the vascular

permeability induced by the antiserum.

In this experiment, the

mixture of antiserum was injected with various concentration of

FXIII.

As shown in Fig. 3, FXIII shows the suppressive effect on

the dye leakage in a dose dependent fashion. We obtained a result

that both 200-fold and 400-fold diluted antiserum exhibit the same

tendency.

Thus the effect of FXIII was examined in 10 guinea pigs

and the dye leakage was measured in extravascular space. As shown

in Fig. 4, FXIII exhibited the suppressive

effect in a dose

dependent manner.

Suppressive effect of FXIII on the swelling

When the antiserum was subcutaneously injected into a dorsal skin

of guinea pig, edema, in addition to hemorrhage was observed at

injection site(20).

Thus we examined the suppressive effect of

FXIII on the swelling.

On injecting the mixture of FXIII and

antiserum, the edema was significantly suppressed by FXIII on day

1 and 2(Fig. 5).

This result indicates that FXIII suppresses the

permeability in the acute and the subacute phase as well.

144

PERMEABILITY

INHIBITION BY FXIII

Vol. 71, No. 2

FIG. 2.

l/800

l/1600

NRS l/50

buffer cont.

Dose response of anti-endothelial

cell antiserum in a

guinea pig. Each sample was injected immediately after a

intravenous dye leakage.

NRS: Normal rabbit serum

DISCUSSION

For more than 20 years after its detection of FXIII, many authors

have reported that a clotting factor, FXIII, influenced a lot of

other systems and thus it was often termed a connective tissue

factors

(21).

The fibrin stabilizing effect is an example of

general properties of this factor which crosslinks proteins with

suitably configurated E - lysyl- and y - glutamyl - residues. Many

kinds of proteins

are listed as substrates

for FXIII,

e.g.

fibrin(l),

collagen(221,

fibronectin(51,

actin(23)

and factor

V(24).

In this context,

the binding

of biogenic

amines to

proteins by FXIII may also participate in the elimination of toxic

substances like histamine.

FXIII concentrate has been recently

used not only for the promotion of the wound healing but also for

the treatment of Shijnlein Henoch Purpura(SHP) (6,101. The clinical

effects of FXIII on SHP are probably due to the stabilization of

microvasculature

leading

to a reduction

of the leakage

at

inflammatory

sites.

Pilger et al(25) has reported that FXIII

shows the suppressive/sealing

effect in a screlodermia patient.

However none of these reports showed the sealing/suppressive

effect on the permeability

by FXIII in animal studies.

This

vasculitis of SHP is regarded as the immunovascular disease that

Vol. 71, No. 2

PERMEABILITY

INHIBITION

BY FXIII

145

the vascular permeability

is enhanced by the formation of the

antigen-antibody

complex not with standing ambiguity of trigger

which may include

drugs,

foods,

insect

bites

or bacterial

infections(ll,12,13,14).

Thus we tried to demonstrate

the

suppressive effect of FXIII on permeability enhancement induced by

anti-endothelial cell antiserum.

As shown in Figs. 1 and 2, anti-

endothelial

cell

antiserum

induces

the

enhancement

of

permeability.

This phenomenon can be caused by factors such as

complement fragments and histamine etc. which are produced by the

activation

of complement

system after complex

formation

of

antiserum with endothelial cells(ll,l2,13,14) .

As this phenomenon

shows the dose dependent manner by antiserum, condition of SHP

patients may be influenced seriously depending on the extent of

the antibody generation.

SHP patients show the increase of plasma

level of IgA and the imbalance

of serum

IgG subclass

and

IgM(13,14,26) .

As shown in Figs. 3,

4 and 5, FXIII suppresses the

vascular permeability

in acute phase and the edema in subacute

phase.

These results are supported by some clinical studies.

Kamitsuji et al.(lO) and Fukui et al.(ll) have reported that FXIII

shows the suppressive effect on the swelling of joints of SHP

patients.

l/ZOO

diluted

/

ted Ab

FIG. 3.

Suppressive

effect

of FXIII

on the

permeability

enhancement induced by anti-endothelial cell antiserum.

FXIII

was used with the final

concentration

at a

injection site of (A), 3.0 U; (B), 1.5 U; (C), 0.75 U;

(D), ^{0.38 U; (E),} medium control.

The mixture of FXIII

and either

200-

or 400-

fold diluted

antiserum

was

injected immediately after the intravenous injection of

dye.

146

PERMEABILITY

INHIBITION BY FXIII

Vol. 71, No. 2

100 -

80 -

% 6o

40 -

20 -

0'

a-GPEC

+1.5U

nFXlll +3.OU

nFXlll

Sample

FIG. 4

Suppressive effect of FXIII on the permeability induced

by

anti-guinea

pig

endothelial

cell

antiserum.

Extraction

of Evans blue at the injection

site was

according to the materials and methods.

n=lO, *: p<o.os,

**: p<O.Ol

In this experiment, we used the 300-fold

diluted anti-endothelial cell antiserum as a permeability

inducer.

FXIII was mixed with antiserum,

then the

mixture was injected intradermally.

2.0

-l

1.8-

0.8 1

0.6

-I

??

T/f

0.0 I

I

I

I

I

0

1

2

3

4

day

FIG. 5.

a-GPEC

+FXIII

Effect of FXIII on the swelling induced by anti-guinea

pig endothelial cell antiserum.

Open circle (0) denotes

the antiserum

alone.

Closed circle

(0) denotes the

FXIII plus antiserum.

n=5, *: pco.05, **: p,O.Ol

Vol. 71, No. 2

PERMEABILITY INHIBITION BY FXIII

147

Pilger et a1.(25) reported that FXIII also shows the suppressive

effect on vascular permeability in sclerodermia patients. These

results suggest that FXIII may crosslink cellular matrices to

prevent the opening of the space between cells(27) and that it may

crosslink the enhancing factors for the permeability(21).

We have

succeeded in demonstrating

the suppressive effect of FXIII on

vascular permeability in an animal study.

This study indicates

that FXIII may play a crucial role in an inflammatory

site.

Consequently it seems that FXIII therapies are necessary for the

treatment of some inflammatory diseases (28,29).

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